NMDA receptors are involved in myriad aspects of CNS function including development, learning and memory but also neurological and psychiatric disorders. Understanding the intricacies of NMDA receptor properties, including permeability and gating, and how different subunit compositions can alter these properties, is critical in developing therapeutic interventions in CNS disorders. To be activated, NMDA receptors require the binding of two glycine and two glutamate molecules (or their equivalents). Several studies indicate that receptors bound by a single glutamate and two glycines do not open, though being singly-bound may facilitate subsequent opening upon a second exposure to glutamate. There are several ways in which NMDA receptors can become singly-bound by glutamate, including exposure to the low levels of ambient glutamate, spillover of glutamate from neighboring synapses and as synaptic receptors sequentially unbind their two glutamates following vesicular release. We propose to study the effects of NMDA receptors bound by two glycines and one glutamate on subsequent activation by a second glutamate binding event. As traditional methods of applying glutamate result in both singly and doubly-bound receptors, the effects of singly-bound receptors are difficult to study directly. Using two recently developed techniques that 1) force expression of NMDA receptors composed of two GluN1 subunits and two different GluN2 subunits and 2) covalently binding only one of the GluN2 subunits to a photoswitchable glutamate ligand, most, if not all, receptors can be forced into the singly-bound state and thus allow the study of a pure population of receptors. Using fast applications of glutamate to outside-out patches (saturating glycine), we will test whether singly-bound NMDA receptors can desensitize, whether the opening of a population of singly-bound receptors is facilitated following succeeding pulses of glutamate and whether the two glutamate binding sites exhibit cooperative binding. In addition, the glutamate unbinding rate from singly-bound receptors will be determined. This is an important parameter as its value determines the period during which a second pulse of glutamate can interact with singly-bound receptors. Together, determination of these properties of NMDA receptors will lead to better predictions about the timing and importance of activation of extrasynaptic receptors and synaptic receptors at neighboring synapses by spillover, as well as how synaptic NMDA receptors just across the synaptic cleft from active release sites respond to repetitive release events.